Cancer often arises when the control of normal cell function goes awry due to defects in critical signal transduction pathways. The signaling pathway regulated by the RasGTPase is one such pathway, and it functions to modulate vital cellular processes, including proliferation, differentiation, survival, and senescence. Members of the Raf serine/threonine kinase family are key intermediates in the Ras pathway, binding directly to activated Ras and serving as the initiating kinases in the ERK cascade, comprised of the Raf, MEK and ERK protein kinases. There are three mammalian Raf proteins, A-Raf, B-Raf, and C-Raf (also known as Raf-1). As might be expected for proteins so centrally involved in cell signaling, the Raf kinases can directly contribute to oncogenic transformation and other human disease states. For example, mutation or amplification of upstream regulators of Raf, such as receptor tyrosine kinases and Ras, frequently results in constitutive signaling through the Raf/MEK/ERK cascade in tumors harboring these alleles. In addition, mutations in the Raf proteins themselves can function as disease drivers. Germline-mutations in C-Raf can be are causative for Noonan and LEOPARD RASopathy syndromes, whereas B-Raf mutations are found in Noonan, LEOPARD, and cardiofaciocutaneous (CFC) RASopathy syndromes, with B-Raf mutations occurring in 75% of CFC patients. Moreover, somatic mutations in B-Raf are observed in 70% of malignant melanomas as well as in many colorectal, ovarian, lung and papillary thyroid carcinomas. Over the years, a major goal of our research team has been to elucidate the mechanisms that regulate Raf catalytic activity. These studies have led to the identification of critical phosphorylation events and protein interactions that contribute to the Raf activation/inactivation cycle. In addition, our work has demonstrated the importance of Raf dimerization in normal and disease-associated signaling and has identified the Raf dimer interface as a therapeutic target. Studies conducted more recently have led to the discovery of a new phospho-regulatory circuit that can suppress Raf activation and Ras signaling under conditions of cellular stress. This circuit functions as a stress-activated signaling checkpoint and can be engaged by cancer therapeutics such as rigosertib, taxol, and vincristine, that activate the JNK/MAPK cascade through oxidative and mitotic stress. During this review period, we developed bioluminescence resonance energy transfer (BRET) technologies to study Raf regulatory interactions under live cell conditions. Using this system, signaling cross-talk between the KIT receptor tyrosine kinase and B-Raf V600E was evaluated, resulting in the discovery that activation of WT-B-Raf by KIT signaling could interfere with melanoma formation driven by B-Raf V600E. In collaboration with the Molecular Targets Laboratory, we have also used the BRET technology to conduct a high-throughput screen to identify natural product compounds that can disrupt or prevent Raf dimerization or the Ras/Raf interaction in live cells. As a result of this effort, new macrophilone-type pyrroloiminoquines were isolated from the marine hydroid Macrorhynchia philippina, and two of the newly identified compounds were found to disrupt Raf dimerization and ERK cascade signaling. These findings indicate that the chemical scaffold of the macrophilones could provide small-molecule therapeutic leads for targeting the ERK cascade in human disease states.